Nirenberg and Leder experiment

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Marshall W. Nirenberg and Philip Leder. The experiment elucidated the triplet nature of the genetic code and allowed the remaining ambiguous codons in the genetic code
to be deciphered.

In this experiment, using a

tRNAs to the ribosome. By associating the tRNA with its specific amino acid
, it was possible to determine the triplet mRNA sequence that coded for each amino acid.

Background

double helix.[1]

In the 1960s, one main DNA mystery scientists needed to figure out was in

amino acids. George Gamow suggested that the genetic code was made of three nucleotides per amino acid. He reasoned that because there are 20 amino acids and only four bases, the coding units could not be single (4 combinations) or pairs (only 16 combinations). Rather, he thought triplets (64 possible combinations) were the coding unit of the genetic code. However, he proposed that the triplets were overlapping and non-degenerate.[2]

Seymour Benzer in the late 1950s had developed an assay using phage mutations which provided the first detailed linearly structured map of a genetic region. Crick felt he could use mutagenesis and genetic recombination phage to further delineate the nature of the genetic code.[3] In the Crick, Brenner et al. experiment, using these phages, the triplet nature of the genetic code was confirmed. They used frameshift mutations and a process called reversions, to add and delete various numbers of nucleotides.[4] When a nucleotide triplet was added to or deleted from the DNA sequence, the encoded protein was minimally affected. Thus, they concluded that the genetic code is a triplet code because it did not cause a frameshift in the reading frame.[5] They correctly concluded that the code is degenerate, that triplets are not overlapping, and that each nucleotide sequence is read from a specific starting point.

Experimental work

The Multi-plater, developed by Leder, helped speed up the process of deciphering the genetic code.[6]

The very first amino acid codon (UUU encoding phenylalanine) was deciphered by Nirenberg and his postdoc

Heinrich Matthaei (see Nirenberg and Matthaei experiment) using long synthetic RNA. However, when similar RNAs are made containing more than one RNA base, the order of the bases was random. For example, a long RNA could be made that had a ratio of C to U of 2:1, and so would contain codons CCU, CUC, UCC at high frequency. When translated by ribosomes, this would produce a protein containing the amino acids proline, leucine, and serine; but it was not possible to say which codon matched which amino acid.[7]

Instead, Nirenberg's group turned to very short synthetic RNAs. They found that the trinucleotide UUU (which is the codon for phenylalanine), was able to cause specific association of phenylalanine-charged tRNA with ribosomes. This association could be detected by passing the mixture through a nitrocellulose filter: the filter captures ribosomes but not free tRNA; however if tRNA was associated with the ribosome, it would also be captured (along with the radioactive phenylalanine attached to the tRNA). They similarly found that trinucleotides AAA or CCC caused ribosome association of lysine-tRNA or proline-tRNA, respectively. [8]

So an experimental plan was clear: synthesize all 64 different trinucleotide combinations, and use the filter assay with tRNAs charged with all 20 amino acids, to see which amino acid associated with which trinucleotide. However, obtaining pure trinucleotides with mixed base sequences, for example GUU, was a daunting challenge. Leder's pioneering studies used trinucleotides made by breaking down long random poly-GU RNA with nuclease and purifying specific trinucleotides by

ribonuclease A in a high concentration of methanol.[11] Nirenberg's postdoc Merton Bernfield used these techniques to determine that UUU and UUC encode phenylalanine, UCU and UCC encode serine, and CCC and CCU encode proline, highlighting a pattern in the way the genetic code redundantly encodes amino acids.[12] Many others in the Nirenberg lab and at NIH contributed to the full decipherment of the genetic code.[11]

Reception and legacy

University of Wisconsin
and Robert W. Holley of the Salk Institute. Working independently, Khorana had mastered the synthesis of nucleic acids, and Holley had discovered the exact chemical structure of transfer-RNA.

The New York Times said of Nirenberg's work that "the science of biology has reached a new frontier," leading to "a revolution far greater in its potential significance than the atomic or hydrogen bomb." Most of the scientific community saw these experiments as highly important and beneficial. However, there were some who were concerned with the new era of molecular genetics. For example, Arne Tiselius, the 1948 Nobel Laureate in Chemistry, asserted that knowledge of the genetic code could "lead to methods of tampering with life, of creating new diseases, of controlling minds, of influencing heredity, even perhaps in certain desired directions."[13]

References

  1. ^ Russell P. (2010). iGenetics: A Molecular Approach, 3rd edition. Pearson/Benjamin Cummings.
  2. ^ Leavitt, Sarah A. (2004). "Deciphering the Genetic Code: Marshall Nirenberg. The Coding Craze". Stetten Museum, Office of NIH History. Archived from the original on 9 February 2020. Retrieved 2009-10-05.
  3. PMID 17350564
    . Retrieved 2018-01-24.
  4. S2CID 4276146
    . Retrieved 2009-10-10.
  5. PMID 14471390.{{cite journal}}: CS1 maint: multiple names: authors list (link
    )
  6. ^ Leavitt, Sarah A. (2004). "Deciphering the Genetic Code: Marshall Nirenberg. Scientific Instruments". Stetten Museum, Office of NIH History. Archived from the original on 9 February 2020. Retrieved 2009-10-05.
  7. ^ Judson H. (1996). The Eighth Day of Creation: Makers of the Revolution in Biology. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
  8. ^
    S2CID 7127820
    .
  9. PMID 14206609
    .
  10. PMID 14243527
    .
  11. ^
    PMID 14729332
    .
  12. PMID 14237203
    .
  13. ^ Fee, E. (2000). "Profiles in Science: The Marshall W. Nirenberg Papers. Public Reaction". National Library of Medicine. Archived from the original on 9 April 2020. Retrieved 9 April 2020.

External links

See also

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